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Membrane depolarization selectively inhibits receptor-operated calcium channels in human T (Jurkat) lymphoblasts
B Sarkadi1, A Tordai, G Gárdos
1National Institute of Haematology and Blood Transfusion, Budapest, Hungary.
Insights
Membrane depolarization selectively inhibits CD3 receptor-mediated calcium influx in Jurkat lymphoblasts, impacting calcium signaling. This finding is crucial for understanding calcium-dependent cell stimulation.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- T-cell activation involves complex signaling pathways, including calcium influx.
- The CD3 membrane antigen plays a critical role in T-cell receptor signaling.
- Understanding the regulation of intracellular calcium is key to deciphering immune cell function.
Purpose of the Study:
- To investigate the relationship between Jurkat lymphoblast membrane potential and calcium signaling.
- To differentiate between calcium release and calcium influx pathways upon CD3 stimulation.
- To determine the effect of membrane potential modulation on CD3-mediated calcium dynamics.
Main Methods:
- Utilized the intracellular fluorescent calcium indicator indo-1 to measure calcium signals.
- Employed the fluorescent dye diS-C3-(5) to estimate cell membrane potential.
- Manipulated membrane potential using K+ concentration, Cl- removal, gramicidin, PCMBS, and valinomycin.
Main Results:
- Membrane depolarization selectively inhibited CD3 receptor-mediated calcium influx, with half-maximum inhibition at -35 to -40 mV.
- Depolarization did not affect stimulus-induced intracellular calcium release.
- Neither depolarization nor hyperpolarization influenced resting calcium influx or basal calcium levels.
Conclusions:
- Membrane potential acts as a significant modulator of the calcium influx pathway in T-cells.
- Selective inhibition of calcium influx by depolarization may regulate calcium-dependent T-cell activation.
- These findings provide insights into the biophysical regulation of immune cell signaling.
Abstract:
Jurkat lymphoblasts were stimulated by a monoclonal antibody against the CD3 membrane antigen and the evoked calcium signal was followed by the intracellular fluorescent calcium indicator indo-1. The technique applied allowed us to separately investigate the stimulus-induced intracellular calcium release and the calcium-influx pathways, respectively. In the same cells membrane potential was estimated by the fluorescent dye diS-C3-(5). The resting membrane potential of Jurkat lymphoblasts under normal conditions was between -55 and -60 mV. Membrane depolarization, obtained by increasing external K+ concentration, removing external Cl-, or by increasing the Na+/K+ leak permeability with gramicidin or PCMBS, did not induce calcium influx in the resting cells and did not influence the CD3 receptor-mediated internal calcium release, while strongly inhibited the receptor-mediated calcium influx pathway. Half-maximum inhibition of this calcium influx was observed at membrane potential values of about -35 to -40 mV and this inhibition did not depend on the external calcium concentration varied between 5 and 2500 microM. Membrane hyperpolarization by valinomycin did not affect either component of the calcium signal. The observed selective inhibition of the receptor-operated calcium influx pathway by membrane depolarization is probably an important modulator of calcium-dependent cell stimulation.